Direct quantitative pcr absent minor groove binders

ABSTRACT

Disclosed herein are methods, compositions and kits for the quantification of a nucleic acid target present on a solid support. This entails quantitative real-time polymerase chain reaction wherein minor groove binders are excluded.

BACKGROUND

Nucleic acid can be quantified using Polymerase Chain Reaction (PCR) bythe detection of amplification products present at the end of PCR,endpoint quantitative PCR, or during PCR, quantitative real-time PCR(qrtPCR). In qrtPCR, fluorescent dyes are generally used to detect PCRproducts during thermal cycling. This allows quantification of thetemplate to be based on the intensity of the fluorescent signal duringthe exponential phase of amplification; before limiting reagents or theinactivation of the polymerase have started to have an effect on theefficiency of PCR amplification.

Specialized instruments are used for qrtPCR assays. These qrtPCRinstruments couple the thermal cycling function of PCR machines with afluorimeter. This combination of a thermal cycling function and afluorimeter allows for in-tube, real-time analysis.

qrtPCR is conducted by placing a reaction tube(s) in a qrtPCR instrumentand subjecting the reaction tube(s) to thermal cycling. During eachthermal cycling round, the reaction tube is illuminated with light andfluorescence emanating from a reaction tube is collected by thefluorimeter. Because the accuracy of qrtPCR depends on the measurementof fluorescent emissions factors that interfere or interrupt the opticalpathway are minimized.

The dominant thinking has been that opaque materials within a qrtPCRreaction tube would mask fluorescent signals and therefore should beexcluded. Counter to this, a few groups have now reported qrtPCR assayswherein filter paper is present in the reaction tube during qrtPCR.Significantly each of these groups has reported that the presence offilter paper increases background fluorescence.

Thus, prior to the instant disclosure a need in the art existed for asimplified qrtPCR assay, wherein the background fluorescent observedwhen filter paper is present is mitigated. The instant disclosure solvesthis problem and more.

BRIEF SUMMARY

The quantization of DNA plays a central role in many applicationsincluding medical diagnostics and forensic DNA analysis. Often DNA usedin these applications is derived from blood or buccal samples applied tofilter paper. Quantification of the DNA then requires the extraction andremoval of the DNA from its source and the filter paper using any one ofa variety of methods. These methods include washing, Chelex® extraction,phenol/chloroform, silica membranes, silica-coated beads, ion exchangemembranes and magnetic beads with an ionic surface. Problems with suchmethods include DNA sample loss and they are laborious.

A workflow which removes the necessity of extraction and removal wouldbe advantageous. Depositing the filter paper into the qrtPCR withoutfirst applying these extraction and removal methods is a solution. Butinclusion of filter paper during qrtPCR affects the baseline fluorescentlevel. Because of this, DNA quantification without extraction andremoval is seldom attempted.

What was not been previously recognized, but is disclosed herein is thata source of the increased levels of background fluorescence is thepresence of a Minor Groove Binder (MGB) in the qrtPCR assay. That is, ina comparison between direct quantification assays where an MGB ispresent to the same assay lacking an MGB, the assay lacking an MGB willhave relatively lower background fluorescence.

Disclosed herein therefore is a method for directly quantifying nucleicacids without prior application of extraction techniques, the methodencompassing depositing a solid support into a reaction vessel,performing a qrtPCR employing a probe while the solid support is withinthe reaction vessel and detecting the level of fluorescence emitted fromthe vessel, wherein a minor groove binder (MGB) is not present in thereaction mixture. In other embodiments a method for directly quantifyingnucleic acids without prior application of extraction techniques isdisclosed, the method encompassing depositing a solid support into areaction vessel, performing a qrtPCR employing a probe without a MGBwhile the solid support is within the reaction vessel and detecting thelevel of fluorescence emitted from the vessel.

In some embodiments, the probe is a 5′-exonuclease probe lacking an MGB.In other embodiments, the method encompasses providing a 5′-exonucleaseprobe lacking an MGB.

In some embodiments, the probe of the method encompasses in a 5′ to 3′order, a target binding region and a tail encompassing a linker and atemplate binding region, wherein the target binding region iscomplementary to an extension product of the probe and wherein an MGB isnot present in the reaction vessel. In other embodiments, the methodencompasses a probe wherein the probe encompasses in a 5′ to 3′ order, atarget binding region and a tail encompassing a linker and a templatebinding region, wherein the target binding region is complementary to anextension product of the probe and wherein the probe lacks an MGB.

In other embodiments, the method encompasses providing a probe whereinthe probe encompasses in a 5′ to 3′ order, a target binding region and atail encompassing a linker and a template binding region, wherein thetarget binding region is complementary to an extension product of theprobe and wherein the probe lacks an MGB.

In some embodiments the solid support is filter paper. In otherembodiments, the method encompasses providing the solid support.

Also disclosed herein is a kit, the kit encompassing a DNA polymerase, a5′-exonuclease probe with a 5′ signaling moiety and a 3′ quenchingmoiety but lacking a MGB, wherein the probe does not form a stablestem-loop structure and a primer pair wherein the primer pair hybridizesand flanks a target sequence found on more than one chromosome.

In some embodiments, the kit encompasses a 5′-exonuclease with a 5′signaling moiety and a 3′ quenching moiety but lacking a MGB. In otherembodiments, the kit encompasses a 5′-exonuclease probe with a 5′quenching moiety and a 3′ signaling moiety lacking an MGB.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of a paper punch on fluorescent detection usinga Quantifiler® Duo quantitative real-time PCR assay. When paper ispresent in the reaction vessel an increase in background fluorescence iswitnessed with all the tested dyes except the ROX™ dye. The increase inbackground fluorescence appears to correlate with the size of the paperpunch. FIG. 1A.—No punch, FIG. 1B.—0.5 mm diameter, FIG. 1C.—1.0 mmdiameter, FIG. 1D.—2.0 mm diameter.

FIG. 2 shows the effect of a paper punch on fluorescent detection usinga Quantifier® Trio quantitative real-time PCR assay. An increase inbackground fluorescence is witnessed in the FAM™ and VIC® dyes but notthe JUN®, ABY® and MUSTANG PURPLE™ dyes when paper is present in thereaction and apparently correlates with the size. FIG. 2A.—No punch,FIG. 2B.—0.5 mm diameter, FIG. 2C.—1.0 mm diameter, FIG. 2D.—2.0 mmdiameter.

FIG. 3 shows the effect of paper punch on background fluorescence withprobes with or without conjugation to a minor groove binder (MGB). FIG.3A) Quantifier® Duo reaction buffer. FIG. 3B) Quantifier® Trio reactionbuffer. Probes conjugated with MGBs showed increased backgroundfluorescent in the presence of 0.5 mm diameter paper punch relative toprobes labeled with the same fluorescent dye but without a conjugatedMGB.

FIG. 4 shows the results of a qrtPCR assay with 1.0 ng. of human malegenomic DNA with or without a paper punch (0.5 mm diameter, 1 mmdiameter and 2 mm diameter) present in the reaction. Backgroundfluorescence was negligible even when paper punches were present in thereaction when the probes were not conjugated to an MGB. FIG. 4A. Probeconjugated with MGB, Y-chromosome target; FIG. 4B. Probe conjugated withMGB, autosomal target; FIG. 4C. Probe without a conjugated MGB,Y-chromosome target; FIG. 4D. Probe without a conjugated MGB, autosomaltarget.

FIG. 5 shows that reduced background fluorescence is independent of theprobe type. 2.5 ng. of human male genomic DNA was assayed with theQuantiplex HYres qrtPCR assay. The Quantiplex HYres utilizes Scoprions®probes for target detection. These probes, also without a conjugatedMGB, demonstrated little background fluorescence with or without a paperpunch (0.5 mm diameter, 1 mm diameter and 2 mm diameter). FIG. 5A.Scorpions® probe without a conjugated MGB, Y-chromosome target; FIG. 5B.Scorpions® probe without a conjugated MGB, autosomal target.

DETAILED DESCRIPTION

Quantitative Real Time Polymerase Chain Reaction (qrtPCR), also referredto as Quantitative Polymerase Chain Reaction (qPCR), is a method basedon PCR which allows for the quantification of a target nucleic acidmolecule during the thermal cycling process. The standard method ofqrtPCR involves suspending a nucleic acid in a liquid, combining thisDNA containing liquid with a reaction mixture and preforming the qrtPCRassay. In a recently describe alternative to this method, a solidsupport with embedded nucleic acid is combined with a reaction mixtureand the qrtPCR assay is conducted while the solid support is present.This alternative methodology has been called direct quantification.

“Direct quantification” refers generally to a method wherein a qrtPCR isconducted while a solid support, such as filter paper, is present in thereaction vessel. The theme driving direct quantification is aminimization in the number or complexity of manipulations necessary toconduct a qrtPCR assay. Thus, direct quantification includes instanceswherein the solid support is not treated before depositing in thereaction vessel. Treatments include the application of extractionmethods, buffers or washing, for example washing with solvent such aswater, prior to depositing the solid support in a reaction vessel forqrtPCR.

Extraction methods include Chelex® extraction, the application ofphenol/chloroform, the application of silica-coated beads, theapplication of ion exchange membranes and the application of magneticbeads with an ionic surface.

An example of a workflow encompassing direct quantification would be theapplication of a specimen, for instance blood or buccal sample, to afilter paper, drying the specimen on the filter paper, excising aportion of the filter paper, for example a disk, and depositing the diskin a reaction vessel for qrtPCR without contacting the dried specimen toa liquid until the qrtPCR assay.

Several groups have reported results from direct quantification assays.Each of these groups observed increased background fluorescence withfilter paper. Taylor reported, for instance, a direct quantificationassay for malarial parasites from blood spotted on 3 MM paper. Taylordetected fluorescence from the general DNA binding dye SYBR® Green toquantify the parasites. Taylor teaches that the filter paper wasresponsible for higher background.

Liu reported a direct quantification assay for total human DNA and humanmale DNA in a sample. Liu used a TaqMan® probe and noted increasedbackground fluorescence associated with the presence of paper in theassay.

Nozawa used direct quantification for quantifying cytomegaloviruspresent in dried urine. Nozawa teaches that nonspecific signals from thedisks interfere with the qrtPCR assay. Nozawa suggests that this isassociated with the type of fluorescent detection system employed;photomultiplier tube or charge-coupled device camera.

Thus, in each reported application of direct quantification it was notedthat increased background fluorescence was the result of, or associatedwith, the presence of paper. Several explanations for the increasedbackground fluorescence associated with the presence of paper includethe fluorescent characteristics of the paper, the paper source andchanges in the orientation of the paper in the reaction well.

A driving assumption for further research was that the physical orchemical properties of the paper were responsible for the observedincrease in background fluorescence. Contrary to expectations, the paperis not a major source for increasing background fluorescence in directquantification assays.

The Quantifiler® Duo and the Quantifiler® Trio are two commerciallyavailable qrtPCR assays largely marketed to forensic scientists. TheQuantifiler® Duo assay quantifies the amount of human DNA and human maleDNA present in a sample. The Quantifiler® Trio assay also quantifies theamount of human DNA and human male DNA present in a sample. A differencebetween the two kits is that whereas the Quantifiler® Duo detects asingle human autosomal target, the Quantifiler® Trio detects twodifferent human autosomal targets. Because the Quantifiler® Trio detectsone more target than the Quantifiler® Duo, the Quantifiler® Trio usesone more dye labeled probe.

Both assays use a probe labeled with a FAM™ dye and a probe labeled witha VIC® dye. The Quantifiler® Duo assay also uses the NED™ dye and theROX™ dye, with the ROX™ dye representing a passive reference control.The Quantifiler® Trio assay differs from the Quantifiler® Duo assay inthat the NED™ dye and the ROX™ dye are not present but the ABY® dye,JUN® dye and Mustang Purple™ dye are. Mustang Purple™ represents thepassive reference control in the Quantifiler® Trio assay.

Both the Quantifiler® Duo and the Quantifiler® Trio assays were testedin direct quantification experiments. In both assays, the presence ofpaper resulted in increased background in the FAM™ dye and the VIC® dyefluorescence channels. In the Quantifiler® Duo assay there was anincrease in the background fluorescence with the NED™ dye labeled probe.The increase in background correlated with larger paper disk size andcycle number (FIG. 1). Because the increased background correlated withthe paper size, this argued strongly for the predominant belief that thepaper was responsible for the increased background.

What these experiments revealed also was that the increased backgroundapparently correlated with wavelength. Table 1 below shows theabsorption and emission maxima of the dyes used in the Quantifiler® Duoand the Quantifiler® Trio assays. Increased background fluorescence wasobserved with FAM™, VIC® and NED™ but not the other dyes (FIGS. 1 and2). These dyes all have an emission maximum below 586 nm. Those dyeswith an emission maximum above 575 nm did not experience increasedbackground; ABY®, ROX™, JUN® and MUSTANG PURPLE™. The data suggeststherefore that the paper is causing increased background by emittingfluorescence in wavelengths below 586 nm.

TABLE 1 ABSORPTION EMISSION DYE λ_(max)/nm λ_(max)/nm FAM ™ 494 518VIC ® 538 554 NED ™ 546 575 ABY ® 568 586 ROX ™ 575 602 JUN ® 606 618MUSTANG PURPLE ™ 633 656

These assays were performed using an Applied Biosystems® 7500 real timePCR instrument. Interestingly, the filters of the Applied Biosystems®7500 real time PCR instrument are configured such that fluorescentsignals from the NED™ dye and the ABY® dye are detected together. If afluorescent property of the paper was responsible for the backgroundthen the expectation would be that both the NED™ dye and the ABY® dyewould have been affected similarly. But since the backgroundfluorescence differs between the two, this argued that something otherthan the general spectral properties of paper was responsible. This leftany number of possible variables to explore.

Minor Groove Binders

A distinction between the probe labeled with the NED™ dye and the probelabeled with ABY® dye was the presence or absence of a minor groovebinder (MGB). “Minor Groove Binder” (MGB) refers to a moiety typicallyhaving a molecular weight of approximately 150 to approximately 2000Daltons. The moiety binds in a non-intercalating manner into the minorgroove of double stranded (or higher order aggregation) DNA, RNA orhybrids thereof, preferably, with an association constant greater thanapproximately 10³M⁻¹.

This type of binding can be detected by established spectrophotometricmethods, such as ultraviolet (u.v.) and nuclear magnetic resonance (nmr)spectroscopy and also by gel electrophoresis. Shifts in u.v. spectraupon binding of a minor groove binder molecule, and nmr spectroscopyutilizing the “Nuclear Overhauser” (NOSEY) effect are particularly wellknown and useful techniques for this purpose. Gel electrophoresisdetects binding of a minor groove binder to double stranded DNA orfragment thereof, because upon such binding the mobility of the doublestranded DNA changes.

Oligonucleotides conjugated with MGBs form unusually stable hybrids withcomplementary DNA. MGBs increase the melting temperature of probes withtheir target sequence, allowing for the design of shorter probes. Theminor groove binder is typically attached to the oligomer through alinker comprising a chain about 20, about 15 atoms, about 10 or about 5atoms.

Because probes utilizing MGBs are relatively shorter they have reducedbackground fluorescence due to decreased distance between a reporter anda quencher. Because of these properties they are widely used in qrtPCR.

Minor groove binding compounds have widely varying chemical structures;however, exemplary minor groove binders have a crescent shape threedimensional structure. Examples of MGBs include certain naturallyoccurring compounds such as netropsin, distamycin and lexitropsin,mithramycin, chromomycin A₃, olivomycin, anthramycin, sibiromycin, aswell as further related antibiotics and synthetic derivatives. Certainbisquarternary ammonium heterocyclic compounds, diarylamidines such aspentamidine, stilbamidine and berenil, CC-1065 and related pyrroloindoleand indole polypeptides, Hoechst 33258, 4′-6-diamidino-2-phenylindole(DAPI) as well as a number of oligopeptides, such as the tripeptide1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxlate (CDPI₃), consistingof naturally occurring or synthetic amino acids are minor groove bindercompounds. Exemplary minor groove binders are described in U.S. Pat. No.6,084,102.

Intercalating moieties or agents are readily distinguished from minorgroove binders on the basis that the intercalating agents are flataromatic (preferably polycyclic) molecules versus the “crescent shape”or analogous geometry of the minor groove binders. An experimentaldistinction can also be made by nmr spectroscopy utilizing the NOSEYeffect.

MGBs are not recognized as being responsible for increasing backgroundfluorescence. Yet, when probes with the same fluorescent label, with andwithout MGBs were tested in the presence of paper, there was drasticdifferences in the levels of background fluorescence (FIG. 3). Thosereactions with MGB had relatively higher background compared to thoselacking MGB.

Accordingly, in some embodiments a method is disclosed, the methodencompassing combining a fluorescently labeled probe and a solidsubstrate in a reaction vessel, preforming a quantitative real-timepolymerase chain reaction and detecting a level of fluorescenceemanating from the reaction vessel during a thermal cycle, wherein aminor groove binder (MGB) is not present in the reaction vessel. In someembodiments, the solid support is untreated.

In some embodiments, a method is disclosed encompassing combining afluorescently labeled probe and a solid substrate in a reaction vessel,preforming a quantitative real-time polymerase chain reaction anddetecting a level of fluorescence emanating from the reaction vesselduring a thermal cycle, wherein the probe is not conjugated to an MGB.

Probes

“Probe” refers to nucleic acid oligonucleotides prepared using a solidsupport. In various embodiments, the probes produce a detectableresponse upon interaction with a target. The probes include at least onedetectable moiety, or a pair of moieties that form an energy transferpair detectable upon some change of state of the probe in response toits interaction with a target.

Oligonucleotides conjugated with MGBs find particular use as probes inqrtPCR assays. Examples of probe based technologies employed in qrtPCRassays include 5′-exonuclease, molecular beacons, hybridization probesand Scorpions® probe.

5′-exonuclease probes, an example of which is a TaqMan™ probe, areoligonucleotides that contain fluorophore and quencher moietiespreferably located on 5′ and 3′ termini. Assays employing 5′-exonucleaseprobes rely on the 5′-exonuclease activity of Taq polymerase to measurethe amount of target sequence in a sample.

During qrtPCR, the complementary strands of a target DNA sequence aremelted apart. When complementary strands of the target DNA are separatea 5′-exonuclease probe, the reverse complement of one of the strands,can hybridize to the target. Polymerase mediated extension of a primeroccurs. When the extending strand reaches the 5′-exonuclease probe, theprobe is degraded. This separates the flourophore from the quencher,resulting in a fluorescent signal. The fluorescent signal isproportional to the amount of target present.

“Complementary” refers to sequence complementarity between two differentnucleic acid strands or between two regions of the same nucleic acidstrand. A first region of a nucleic acid is complementary to a secondregion of the same or a different nucleic acid if, when the two regionsare arranged in an anti-parallel fashion, at least one nucleotideresidue of the first region is capable of base pairing (that is,hydrogen bonding) with a residue of the second region, thus forming ahydrogen-bonded duplex.

“Substantially complementary” refers to two nucleic acid strands (forexample, a strand of a target nucleic acid and a complementarysingle-stranded oligonucleotide probe) that are capable of base pairingwith one another to form a stable hydrogen-bonded duplex under stringenthybridization conditions. In general, “substantially complementary”refers to two nucleic acids having at least 70%, for example, about 75%,80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% complementarity.

In some embodiments a method is disclosed, the method encompassingcombining a fluorescently labeled probe and a solid substrate in areaction vessel, preforming a quantitative real-time polymerase chainreaction and detecting a level of fluorescence emanating from thereaction vessel during a thermal cycle while the solid support ispresent in the reaction vessel, wherein a fluorophore is present at the5′ terminus of the probe and a quencher is present at the 3′ terminus ofthe probe and wherein a minor groove binder (MGB) is not present in thereaction vessel. In some embodiments, a quencher is present at the 5′terminus of the probe and a fluorophore is present at the 3′ terminus ofthe probe. In other embodiments, the solid support is untreated.

In other embodiments a method is disclosed, the method encompassingcombining a fluorescently labeled probe and a solid support in areaction vessel, preforming a quantitative real-time polymerase chainreaction and detecting a level of fluorescence emanating from thereaction vessel during a thermal cycle while the solid support ispresent in the reaction vessel, wherein a fluorophore is present at the5′ terminus of the probe and a quencher is present at the 3′ terminus ofthe probe and wherein a minor groove binder (MGB) is not conjugate tothe probe. In some embodiments, the MGB is not conjugated to the 3′terminus of the probe. In other embodiments, the MGB is not conjugatedto the 5′ terminus of the probe. In other embodiments, the solid supportis untreated.

In other embodiments, a method is disclosed encompassing providing afluorescently labeled probe, combining the fluorescently labeled probeand a solid support in a reaction vessel, preforming a quantitativereal-time polymerase chain reaction and detecting a level offluorescence emanating from the reaction vessel during a thermal cyclewhile the solid support is present in the reaction vessel, wherein afluorophore is present at the 5′ terminus of the probe and a quencher ispresent at the 3′ terminus of the probe and wherein a minor groovebinder (MGB) is not present in the reaction vessel. In otherembodiments, the solid support is untreated.

In other embodiments a method is disclosed, the method encompassingproviding a fluorescently labeled probe, combining the fluorescentlylabeled probe and a solid substrate in a reaction vessel, preforming aquantitative real-time polymerase chain reaction and detecting a levelof fluorescence emanating from the reaction vessel during a thermalcycle while the solid support is present in the reaction vessel, whereina fluorophore is present at the 5′ terminus of the probe and a quencheris present at the 3′ terminus of the probe and wherein a minor groovebinder (MGB) is not conjugate to the probe. In some embodiments, the MGBis not conjugated to the 3′ terminus of the probe. In other embodiments,the MGB is not conjugated to the 5′ terminus of the probe. In otherembodiments, the solid support is untreated.

“Fluorophore” refers to a moiety that is inherently fluorescent ordemonstrates a change in fluorescence upon binding to a biologicalcompound or metal ion, or when metabolized by an enzyme. Numerousfluorophores are known, examples of which include coumarins, acridines,fUrans, dansyls, cyanines, pyrenes, naphthalenes, benzofurans,quinolines, quinazolinones, indoles, benzazoles, borapolyazaindacenes,oxazines and xanthenes, with the latter including fluoresceins,rhodamines, rosamines and rhodols.

A number of fluorescent dyes can be detected in a qrtPCR assay and caninclude, without limitation, the following: 5- or 6-carboxyfluorescein(FAM™), VIC™, NED™, fluorescein, fluorescein isothiocyanate (FITC),IRD-700/800, cyanine dyes, such as CY3™, CY5™, CY3.5™, CY5.5™, Cy7™,xanthen, 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX™),6-carboxy-1,4-dichloro-2′,7′-dichloro-fluorescein (TET®),6-carboxy-4′,5′-dichloro-2′,7′-dimethodyfluorescein (JOE™),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA™), 6-carboxy-X-rhodamine(ROX™), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6),rhodamine, rhodamine green, rhodamine red, rhodamine 110, Rhodamin 6G®,BODIPY dyes, such as BODIPY TMR, oregon green, coumarines, such asumbelliferone, benzimides, such as Hoechst 33258; phenanthridines, suchas Texas Red®, California Red®, Yakima Yellow, Alexa Fluor® 350, AlexaFluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 500, AlexaFluor® 514, Alexa Fluor®532, Alexa Fluor® 546, Alexa Fluor® 555, AlexaFluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor® 633, AlexaFluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, AlexaFluor® 750, PET®, ethidium bromide, acridinium dyes, carbazol dyes,phenoxazine dyes, porphyrine dyes, polymethin dyes, Atto 390, Atto 425,Atto 465, Atto 488, Atto 495, Atto 520, Atto 532, Atto 550, Atto 565,Atto 590, Atto 594, Atto 620, Atto 633, Atto 647N, Atto 655, Atto RhoG6,Atto Rhol 1, Atto Rhol2, Atto Rho101, BMN™-5, BMN™-6, CEQ8000 D2,CEQ8000 D3, CEQ8000 D4, DY-480XL, DY-485XL, DY-495, DY-505, DY-510XL,DY-521XL, DY-521XL, DY-530, DY-547, DY-550, DY-555, DY-610, DY-615,DY-630, DY-631, DY-633, DY-635, DY-647, DY-651, DY-675, DY-676, DY-680,DY-681, DY-700, DY-701, DY-730, DY-731, DY-732, DY-750, DY-751, DY-776,DY-780, DY-781, DY-782, CAL Fluor® Gold 540, CAL Fluor RED 590, CALFluor Red 610, CAL Fluor Red 635, IRDye® 700Dx, IRDye® 800CW, MarinaBlue®, Pacific Blue®, Yakima Yellow®,6-(4,7-Dichloro-2′,7′-diphenyl-3′,6′-dipivaloylfluorescein-6-carboxamido)-hexyl-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite(SIMA), CAL Fluor® Gold 540, CAL Fluor® Orange 560, CAL Fluor Red 635,Quasar® 570, Quasar® 670, LIZ, Sunnyvale Red, LC Red® 610, LC Red® 640,LC Red®670 and LC Red®705.

“Quencher” refers to any fluorescent-modifying moiety that can attenuateat least partly the light emitted by a fluorophore. This attenuation isreferred to as “quenching.” The excitation of a fluorophore in thepresence of the quenching group leads to an emission signal that is lessintense than expected, or even completely absent. Quenching typicallyoccurs through energy transfer between the excited fluorophore and thequenching group.

Very little fluorescence is emitted from intact 5′-exonuclease probe dueto efficient intra-molecular quenching. However, during PCRamplification, the probe specifically hybridizes to its target sequenceand the 5′-3′-exonuclease activity of Taq polymerase cleaves the probebetween fluorophore and quencher moieties. “Hybridize” refers to twonucleic acid strands associated with each other which may or may not befully base-paired.

Molecular beacons are single-stranded oligonucleotide probes that arenon-fluorescent in isolation, but become fluorescent upon hybridizationto target sequences. Non-hybridized molecular beacons form stem-loopstructures, possessing a fluorophore covalently linked to one end of themolecule and a quencher linked to the other, such that the hairpin ofthe beacon places the fluorophore moiety in close proximity with thequencher. When molecular beacons hybridize to target sequences,fluorophore and quencher moieties become spatially separated, such thatthe fluorophore is no longer quenched and the molecular beaconfluoresces. The secondary structure of the molecular beacon conveys highspecificity to the hybridization probe.

Accordingly, in some embodiments a method is disclosed, the methodencompassing combining a fluorescently labeled probe and a solidsubstrate in a reaction vessel, preforming a quantitative real-timepolymerase chain reaction and detecting a level of fluorescenceemanating from the reaction vessel during a thermal cycle while thesolid support is present in the reaction vessel, wherein the probepossesses a self-complementary sequence and wherein a minor groovebinder (MGB) is not present in the reaction vessel. In some embodiments,the self-complementary sequence forms a hairpin. In other embodiments,the solid support is untreated.

In some embodiments, the method encompasses combining a fluorescentlylabeled probe and a solid substrate in a reaction vessel, preforming aquantitative real-time polymerase chain reaction and detecting a levelof fluorescence emanating from the reaction vessel during a thermalcycle while the solid support is present in the reaction vessel, whereinthe probe possesses a self-complementary sequence and wherein a minorgroove binder (MGB) is not conjugated to the probe. In some embodiments,the self-complementary sequence of the probe forms a hairpin and an MGBis not conjugated to the probe. In other embodiments, the solid supportis untreated.

In some embodiments, the method encompasses providing a fluorescentlylabeled probe, wherein the probe possesses a self-complementary sequenceand wherein an MGB is not conjugated to the probe. In some embodiments,method encompasses providing a probe possessing a self-complementarysequence wherein the self-complementary sequence of the probe forms ahairpin and an MGB is not conjugated to the probe.

Hybridization probes are oligonucleotides that are singly labeled with afluorophore moiety. Two such oligonucleotides are required for eachhybridization probe assay, one labeled with a donor fluorophore and theother with an acceptor fluorophore. Fluorescein is commonly employed asthe donor and Cy5™, LC Red® 640 and LC Red®705 are commonly used asacceptors. Excitation of the donor fluorophore produces an emissionspectrum that overlaps with the absorption spectrum of the acceptorfluorophore. Hybridization probe pairs are designed to recognizeadjacent nucleotide sequences within target molecules. In isolation, theacceptor oligonucleotide is not excited and does not generate afluorescent signal. However, during hybridization to polynucleotidetarget sequences, the donor and acceptor probes are brought into closeproximity, allowing fluorescence resonance energy transfer from thedonor to the acceptor. Fluorescent signal from the acceptor fluorophoreis only emitted when both probes are hybridized to the target molecule.When incorporated into PCR reactions, fluorescence from the acceptorprobe is monitored once per cycle of amplification, to facilitatereal-time measurement of product accumulation, where the amount offluorescence emitted by the acceptor is proportional to the quantity oftarget synthesized.

In some embodiments, the method encompasses combining a probe labeledwith a donor fluorophore, a probe labeled with an acceptor fluorophoreand a solid substrate in a reaction vessel, preforming a quantitativereal-time polymerase chain reaction and detecting a level offluorescence emanating from the reaction vessel during a thermal cyclewhile the solid support is present in the reaction vessel and wherein anMGB is not present in the reaction vessel. In other embodiments, thesolid support is untreated.

In other embodiments, the method encompasses combining a probe labeledwith a donor fluorophore, a probe labeled with an acceptor fluorophoreand a solid substrate in a reaction vessel, preforming a quantitativereal-time polymerase chain reaction and detecting a level offluorescence emanating from the reaction vessel during a thermal cyclewhile the solid support is present in the reaction vessel, wherein aminor groove binder (MGB) is not conjugated to either probe. In someembodiments, an MGB is conjugated to the probe labeled with a donorfluorophore. In other embodiments, an MGB is conjugated to the probelabeled with an acceptor fluorophore. In other embodiments, the solidsupport is untreated.

In some embodiments, the method encompasses providing a probe labeledwith a donor fluorophore, combining the probe labeled with the donorfluorophore, a probe labeled with an acceptor fluorophore and a solidsubstrate in a reaction vessel, preforming a quantitative real-timepolymerase chain reaction and detecting a level of fluorescenceemanating from the reaction vessel during a thermal cycle while thesolid support is present in the reaction vessel, wherein a minor groovebinder (MGB) is not conjugated to either probe.

In other embodiments, the method encompasses providing a probe labeledwith an acceptor fluorophore, combining a probe labeled with a donorfluorophore, the probe labeled with an acceptor fluorophore and a solidsubstrate in a reaction vessel while the solid support is present in thereaction vessel, preforming a quantitative real-time polymerase chainreaction and detecting a level of fluorescence emanating from thereaction vessel during a thermal cycle, wherein a minor groove binder(MGB) is not conjugated to either probe. In some embodiments, the methodencompasses providing a probe labeled with an acceptor fluorophoreconjugated with an MGB. In other embodiments, the method encompassesproviding a probe labeled with a donor fluorophore conjugated with anMGB.

5′-exonuclease, molecular beacon and hybridization probe assays arebimolecular systems that have the probe and target sequences located onseparate DNA strands. Scorpions® probes operate through single molecularbinding events, where the probe and amplified target sequence arelocated on the same DNA strand. Single molecular binding events arekinetically favored over bimolecular hybridization. Scorpions® probesencompass a primer with an attached probe tail sequence, where the probesequence is contained within a stem-loop secondary structure similar tothat of a molecular beacon. In the non-extended form, Scorpions® primersare non-fluorescent due to fluorophore and quencher moieties being inclose proximity. During PCR, the primer component of the Scorpions®probe is extended at its 3′ end producing the homologous target sequencerequired for probe hybridization. When the Scorpions® probe sequencehybridizes to amplified target the fluorophore and quencher moietiesbecome spatially separated generating significant increases influorescent signal concurrent with target amplification.

Accordingly, in some embodiments a method is disclosed, the methodencompassing combining a fluorescently labeled probe and a solidsubstrate in a reaction vessel and preforming a quantitative real-timepolymerase chain reaction and detecting a level of fluorescenceemanating from the reaction vessel during a thermal cycle while thesolid support is present in the reaction vessel, wherein the probecomprises in a 5′ to 3′ order, a target binding region and a tailcomprising a linker and a template binding region, wherein the targetbinding region is complementary to an extension product of the probe andwherein a minor groove binder is not present in the reaction vessel. Inother embodiments, the solid support is untreated.

When using a Scorpions® probe the “template” is the underlining nucleicacid sought to be quantified in a qrtPCR.

In other embodiments, the method encompasses combining a fluorescentlylabeled probe and a solid substrate in a reaction vessel and preforminga quantitative real-time polymerase chain reaction and detecting a levelof fluorescence emanating from the reaction vessel during a thermalcycle while the solid support is present in the reaction vessel, whereinthe probe comprises in a 5′ to 3′ order, a target binding region and atail comprising a linker and a template binding region, wherein thetarget binding region is complementary to an extension product of theprobe and wherein the probe is not conjugated with a minor groove binder(MGB). In other embodiments, the solid support is untreated.

In some embodiments, the method encompasses providing a probe, whereinthe probe comprises in a 5′ to 3′ order, a target binding region and atail comprising a linker and a template binding region, wherein thetarget binding region is complementary to an extension product of theprobe, combining a fluorescently labeled probe and a solid substrate ina reaction vessel and preforming a quantitative real-time polymerasechain reaction and detecting a level of fluorescence emanating from thereaction vessel during a thermal cycle while the solid support ispresent in the reaction vessel and wherein a minor groove binder is notpresent in the reaction vessel.

In some embodiments, the method encompasses providing a probe, whereinthe probe comprises in a 5′ to 3′ order, a target binding region and atail comprising a linker and a template binding region, wherein thetarget binding region is complementary to an extension product of theprobe, combining a fluorescently labeled probe and a solid substrate ina reaction vessel and preforming a quantitative real-time polymerasechain reaction and detecting a level of fluorescence emanating from thereaction vessel during a thermal cycle while the solid support ispresent in the reaction vessel and wherein the probe is not conjugatedwith a minor groove binder.

Instruments

The fluorescent signal emitted from the vessel during the qrtPCR can bedetected using a number of different types of detectors includingCharge-coupled Device (CCD), photodiode and photomultiplier tube. A CCDconverts the light that it captures into digital data. The quality ofthe image captured is determined by the resolution, usually expressed interms of megapixels. CCDs are typically used to capture an image of avessel or reaction plate, whose content is then interpreted byinstrument software.

A photodiode is a type of photodetector that, when exposed to light,causes a current to flow. A photomultiplier tube multiplies the currentthat is produced by incident light.

Accordingly, in some embodiments a method is disclosed, the methodencompassing combining a fluorescently labeled probe and a solidsubstrate in a reaction vessel, preforming a quantitative real-timepolymerase chain reaction and detecting a level of fluorescenceemanating from the reaction vessel during a thermal cycle while thesolid support is present in the reaction vessel, wherein thefluorescence is detected with a charged-coupled device and wherein aminor groove binder (MGB) is not present in the reaction vessel. Inother embodiments, the fluorescence is detected with a photodiode. Insome embodiments, the fluorescence is detected with a photomultiplier.In some embodiments, one or more probes in the reaction vessel are notconjugated with a MGB.

In some embodiments, fluorescence is detected and the quantity of atarget is thereby determined.

In some embodiments, the method encompasses providing the qrtPCRinstrument.

Samples

In some embodiments a method is disclosed, the method encompassingproviding a solid support contacted to a surface, combining afluorescently labeled probe and the solid support in a reaction vessel,preforming a quantitative real-time polymerase chain reaction anddetecting a level of fluorescence emanating from the reaction vesselduring a thermal cycle while the solid support is present in thereaction vessel, wherein a minor groove binder (MGB) is not present inthe reaction vessel. In other embodiments, the method encompassesproviding a solid support contacted to a surface, combining afluorescently labeled probe and the solid support in a reaction vessel,preforming a quantitative real-time polymerase chain reaction anddetecting a level of fluorescence emanating from the reaction vesselduring a thermal cycle while the solid support is present in thereaction vessel, wherein the probe is not conjugated with a minor groovebinder (MGB).

In some embodiments, the method encompasses combining a fluorescentlylabeled probe and the solid support in a reaction vessel, wherein abiological sample has been applied to the solid support, preforming aquantitative real-time polymerase chain reaction and detecting a levelof fluorescence emanating from the reaction vessel during a thermalcycle while the solid support is present in the reaction vessel, whereina minor groove binder (MGB) is not present in the reaction vessel. Inother embodiments, the method encompasses combining a fluorescentlylabeled probe and the solid support in a reaction vessel, wherein abiological sample has been applied to the solid support, preforming aquantitative real-time polymerase chain reaction and detecting a levelof fluorescence emanating from the reaction vessel during a thermalcycle while the solid support is present in the reaction vessel, whereinthe probe is not conjugated with a minor groove binder (MGB). In otherembodiments, the solid support is untreated.

In some embodiments, the surface contacted is suspected of having abiological sample or a specimen. In some embodiments, the solid supportis contacted to a surface during the course of a criminal investigation.

A “biological sample” refers to a collection made from an organism suchas a eukaryote, prokaryote or virus. In some embodiments, the biologicalsample is not a malarial parasite.

In some embodiments, the method encompasses combining a fluorescentlylabeled probe and the solid support in a reaction vessel, wherein aspecimen has been applied to the solid support, preforming aquantitative real-time polymerase chain reaction and detecting a levelof fluorescence emanating from the reaction vessel during a thermalcycle while the solid support is present in the reaction vessel, whereina minor groove binder (MGB) is not present in the reaction vessel. Inother embodiments, the method encompasses combining a fluorescentlylabeled probe and the solid support in a reaction vessel, wherein aspecimen has been applied to the solid support, preforming aquantitative real-time polymerase chain reaction and detecting a levelof fluorescence emanating from the reaction vessel during a thermalcycle while the solid support is present in the reaction vessel, whereinthe probe is not conjugated with a minor groove binder (MGB). In otherembodiments, the solid support is untreated.

A “specimen” refers to whole blood, plasma, serum, saliva, buccalsample, sweat, vaginal secretions, semen, tissues, urine, cerebrospinalfluid and touch nucleic acid. “Touch nucleic acid” or “transfer nucleicacid” refers to nucleic acid that may be left on a surface after beingcontacted by an organism. For example, a fingerprint can contain nucleicacid and represents a touch nucleic acid. In some embodiments, thespecimen is no urine. In some embodiments, the specimen is not from achild less than a month old.

In some embodiments, the method encompasses combining a fluorescentlylabeled probe, a primer or a primer pair and a solid support in areaction vessel, preforming a quantitative real-time polymerase chainreaction and detecting a level of fluorescence emanating from thereaction vessel during a thermal cycle while the solid support ispresent in the reaction vessel, wherein a minor groove binder (MGB) isnot present in the reaction vessel. In other embodiments, a method isdisclosed encompassing combining a fluorescently labeled probe, a primeror a primer pair and a solid support in a reaction vessel, preforming aquantitative real-time polymerase chain reaction and detecting a levelof fluorescence emanating from the reaction vessel during a thermalcycle while the solid support is present in the reaction vessel, whereinthe probe is not conjugated with a minor groove binder (MGB). In otherembodiments, the solid support is untreated.

In some embodiments, the method encompasses providing a primer or aprimer pair, combining a fluorescently labeled probe, the primer or theprimer pair and a solid support in a reaction vessel, preforming aquantitative real-time polymerase chain reaction and detecting a levelof fluorescence emanating from the reaction vessel during a thermalcycle while the solid support is present in the reaction vessel, whereina minor groove binder (MGB) is not present in the reaction vessel. Inother embodiments, the method encompasses providing a primer or a primerpair, combining a fluorescently labeled probe, the primer or the primerpair and a solid support in a reaction vessel, preforming a quantitativereal-time polymerase chain reaction and detecting a level offluorescence emanating from the reaction vessel during a thermal cyclewhile the solid support is present in the reaction vessel, wherein theprobe is not conjugated with a minor groove binder (MGB). In otherembodiments, the solid support is untreated.

“Primer(s)” refer to isolated oligonucleotides that can anneal to acomplementary nucleic acid strand and can be extended, for example by apolymerase. A primer pair refers to two primers that anneal to oppositestrands of a DNA target. Primers flank a target.

“Target” refers to a nucleic acid sequence to be detected. Copies of thetarget sequence which are generated during the amplification reactionare referred to as amplification products, amplimers, or amplicons. Atarget nucleic acid may be composed of segments of a chromosome, acomplete gene with or without intergenic sequence, segments or portionsof a gene, with or without intergenic sequence. Target nucleic acids mayinclude a wild-type sequence(s), a mutation, deletion or duplication,tandem repeat regions, a gene of interest, a region of a gene ofinterest or any upstream or downstream region thereof. Target nucleicacids may represent alternative sequences or alleles of a particulargene. Target nucleic acids may be derived from genomic DNA, cDNA, orRNA. A target nucleic acid may be DNA or RNA from a eukaryotic cell or anucleic acid copied or amplified therefrom but not a prokaryotic cell orvirus. A target nucleic acid may be DNA or RNA from a prokaryotic cellor a nucleic acid copied or amplified therefrom but not a eukaryoticcell or virus. A target nucleic acid may be DNA or RNA from a virus butnot a eukaryotic cell or prokaryotic cell.

In some embodiments, the target is a multicopy locus. A multicopy locusis not a repetitive element and has copies on at least two differentchromosomes, for example chr. 1 and chr. 2 and chr. X and chr. Y. Twodifferent chromosomes do not refer to one chromosome inherited from themother and the other from the father. In some embodiments, the multicopylocus is one with 5-50 copies in the genome. In other embodiments, themulticopy locus is one with 10-40 copies in the genome. In someembodiments, the multicopy locus is one with 10-25 copies in the genome.In other embodiments, the multicopy locus is one with 13-20 copies inthe genome. In some embodiments, the multicopy locus is one with 14-19copies in the genome.

Accordingly, in some embodiments a method is disclosed, the methodencompassing combining a fluorescently labeled probe and a solid supportin a reaction vessel, preforming a quantitative real-time polymerasechain reaction and detecting a level of fluorescence emanating from thereaction vessel during a thermal cycle while the solid support ispresent in the reaction vessel, wherein a minor groove binder (MGB) isnot present in the reaction vessel and a target is a multicopy locus. Inother embodiments, a method is disclosed, the method encompassingcombining a fluorescently labeled probe and a solid support in areaction vessel, preforming a quantitative real-time polymerase chainreaction and detecting a level of fluorescence emanating from thereaction vessel during a thermal cycle while the solid support ispresent in the reaction vessel, wherein a minor groove binder (MGB) isnot conjugated to the probe and a target is a multicopy locus. In otherembodiments, the solid support is untreated.

In some embodiments, the disclosed method encompasses providing afluorescently labeled probe, wherein the probe detects a multicopylocus. In other embodiments, the disclosed method encompasses providinga primer or a primer pair wherein the primer or the primer pair flanks amulticopy locus.

“Multiplex PCR” refers to the simultaneous amplification of more thanone target polynucleotide in a vessel. In some embodiments, at least 2targets are amplified simultaneously. In some embodiments, at least 3targets are amplified simultaneously. In other embodiments, at least 4targets are amplified simultaneously. In still other embodiments, atleast 5 targets are amplified simultaneously. In other embodiments, atleast 6 targets are amplified simultaneously. In some embodiments, 7 ormore targets are amplified simultaneously.

Solid Support

“Paper” refers to sheet-like masses and molded products containingcellulosic fibers. Cellulosic fibers can include digested fibers fromsoftwood (derived from coniferous trees), hardwood (derived fromdeciduous trees) or cotton linters. Fibers from Esparto grass, bagasse,kemp, flax, and other lignaceous and cellulosic fiber sources may alsobe utilized.

In some embodiments, the filter paper is Whatman 903. In someembodiments, the filter paper is Ahlstrom grade 226. In someembodiments, the filter paper is Munktell TFN. In some embodiments, thefilter paper is Isocode.

In some embodiments, a weak base is sorbed to the filter paper beforedepositing the filter paper into the vessel. A “weak base” refers to abase which has a pH of about 6 to 10, preferably about pH 8 to 9.5. Onefunction of the weak base may be to act as a buffer to maintain acomposition pH of about 6 to 10, preferably about pH 8.0 to 9.5, forexample, pH 8.6. Hence, a weak base suitable may, in conjunction withother components, provide a pH of 6 to 10, preferably, about pH 8.0 to9.5. Weak bases include organic and inorganic bases. Examples ofinorganic weak bases include, for example, an alkali metal carbonate,bicarbonate, phosphate or borate (For example, sodium, lithium, orpotassium carbonate). Organic weak bases include, for example,tris-hydroxymethyl amino methane (Tris), ethanolamine, triethanolamineand glycine and alkaline salts of organic acids (for example, trisodiumcitrate). The weak base may be either a free base or a salt, forexample, a carbonate salt.

In some embodiments, a chelating agent is sorbed to the filter paperbefore depositing the filter paper into the vessel. A “chelating agent”refers any compound capable of complexing multivalent ions includingGroup II and Group III multivalent metal ions and transition metal ions(for example, Cu, Fe, Zn, Mn, etc). Ethylene diamine tetraacetic acid(EDTA) is an example of a chelating agent. Chelating agents such as acitrate or oxalate can also be applied to the filter paper.

In some embodiments, a detergent is sorbed to the filter paper beforedepositing the filter paper into the vessel. “Detergent” includes ionicdetergents, preferably anionic detergents. A preferred anionic detergentmay have a hydrocarbon moiety, such as an aliphatic or aromatic moiety,and one or more anionic groups. Particularly preferred anionicdetergents include sodium dodecyl sulphate (SDS) and sodium laurylsarcosinate (SLS).

In some embodiments, a weak base, a chelating agent and a detergent aresorbed to the filter paper before depositing the filter paper into thevessel. In some embodiments, the filter paper is FTA™. In someembodiments, a method is provided encompassing providing a filter paperwherein a weak base, a chelating agent and a detergent have been sorbedto the filter; for example, providing FTA™ paper.

A “non-cellulosic fiber” refers to a polymeric material characterized byhaving hydroxyl groups attached to the polymer backbone, for exampleglass fibers and synthetic fibers modified with hydroxyl groups. Otherfibrous materials include synthetic fibers, such as rayon, polyethyleneand polypropylene can also be utilized in combination with naturalcellulosic fibers or other fibers containing hydroxyl groups. Mixturesof any of the foregoing fibers may be used.

In some embodiments the solid support is opaque. In other embodiments,the solid support is clear or such that approximately 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, 99% or more of the light shined on the solid support passesthrough it.

Kits

Disclosed herein are kits of components. In some embodiments, thecomponents of the kit encompass a primer and a fluorescently labeledprobe with a 5′ signaling moiety and a 3′ quencher, wherein the probedoes not form a stable stem loop structure and the probe is notconjugated with a MGB. In other embodiments, the components of the kitencompass a primer and two or more fluorescently labeled probes with a5′ signaling moiety and a 3′ quencher, wherein the probes do not formstable stem loop structures and the probes are not conjugated with aMGB. In some embodiments, the probe or probes detect a multicopy target.

In some embodiments, the components of the kit encompasses at least twoprimers a fluorescently labeled probe with a 5′ signaling moiety and a3′ quencher, wherein the probe does not form a stable stem loopstructure and the probe is not conjugated with a MGB. In otherembodiments, the components of the kit encompass at least two primersand two or more fluorescently labeled probes with a 5′ signaling moietyand a 3′ quencher, wherein the probes do not form stable stem loopstructures and the probes are not conjugated with a MGB. In someembodiments, two primers of the at least two primers form a primer pair.In other embodiments, the one or more primers are complementary to asequence flanking a multicopy target locus. In some of the embodiments,none of the probes contained in the kit are conjugated with an MGB. Insome of the embodiments, the kit encompasses a plurality of probes andnone of the probes contained in the kit are conjugated with an MGB.

In some embodiments, a kit of components is disclosed wherein the kit ofcomponents encompasses a multiplicity of primers and a multiplicity offluorescently labeled probes, wherein each of the probes possesses a 5′signaling moiety and a 3′ quencher, wherein each of the probes detects amulticopy locus and wherein each probe is not conjugated with an MGB andwherein each of the multiplicity of primers is complementary to asequence flanking a multicopy locus.

In some embodiments, the components of the kit encompass a polymerase.

A stable stem-loop is one with a melting temperature above 40° C., orabove 45° C., or above 50° C., or above 55° C., or above 60° C. or above65° C., or above 70° C., or above 75° C. or above 80° C. or more.

In some embodiments, one or more of the components of the kit are in oneor more vessels. In some embodiments, the kit is packaged in a singleenclosure, for instance a box. In some embodiments, the reagents areprovided in vessels of suitable strength for direct use or use afterdilution.

Disclosed herein are compositions, the compositions being thecombination of constituents of the methods, and the intermediates of themethods disclosed above or the end product of the methods disclosedabove.

Accordingly, in some embodiments a composition is disclosed thecomposition encompassing a fluorescently labeled probe, a primer and asolid support, wherein the fluorescently labeled probe is not conjugatedwith an MGB. In some embodiments, the composition encompasses two ormore fluorescently labeled probes, two or more primers and a solidsupport, wherein each of the two or more fluorescently labeled probesare not conjugated with an MGB. In some embodiments, the compositionencompasses two or more fluorescently labeled probes, two or moreprimers and a solid support, wherein the composition does not encompassan MGB. In some embodiments, one or more probes detect a multicopylocus. In some embodiments, the one or more primers flank a multicopylocus.

Examples The Effect of Punch Size and Baseline Setting on qrtPCRQuantification

Because illuminating the sample with light and detecting thefluorescence signal is central to a qrtPCR assay the influence of paperpunch size on the qrtPCR assay was tested. Punches of various diameters,0.5 mm, 1 mm and 2 mm were tested in a qrtPCR assay. Individual puncheswith different diameters were generated from PE-swabs and placeddirectly into a well of MicroAmp® Optical 96-Well Reaction Plate.Punches were made from negative control PE-swabs; that is, PE-swabs thathad not be used to swab a surface. The punches were then subjected toqrtPCR analysis.

To analyze the influence of punches and punch size on qrtPCR, punches ofvarious sizes were deposited in wells of a MicroAmp® Optical 96-WellReaction Plate. Wells without punches were also analyzed. To each wellwere added 10.5 μL Quantifiler® Duo Primer Mix, 12.5 μL Quantifiler® DuoPCR reaction mix and 2 μL de-ionized water. Also present in the wellswas the IPC template control. Quantification reactions were carried outon Applied Biosystems 7500 Real-Time PCR System using the manufacturerecommended protocol. The Applied Biosystems 7500 Real-Time PCR Systemutilizes a CCD detector. The quantification results were analyzed usingSDS Software v2.0.6 (Life Technologies). Results from this analysis areshown in FIG. 1.

Except for the ROX™ passive reference fluorescent channel, the presenceof a filter paper punch in the reaction well results in elevatedbackground florescent signal in the FAM™, VIC® and NED™ fluorescentchannels. The magnitude of the background elevation is correlated withthe size of the filter paper punch in the reaction well. In addition, itwas also observed that the background florescent signal increases aftereach thermal cycle and the rate increase is apparently correlated withthe size of the filter paper punch.

The Quantifiler® Trio is a recently developed kit for quantifying humanDNA in a sample. While the Quantifiler® Duo detects the presence of twohuman targets, an autosomal target and a Y-chromosome target, theQuantifiler® Trio detects two autosomal targets and a Y-chromosomaltarget. As described above with the Quantifiler® Duo, the influence ofpunches and punch size using the Quantifiler® Trio was also tested.

Punches of various diameters, 0.5 mm, 1 mm and 2 mm were made anddeposited in wells of a MicroAmp® Optical 96-Well Reaction Plate. Wellswithout punches were also analyzed. To each well were added 10.5 μLQuantifiler® Trio Primer Mix, 12.5 μL Quantifiler® Trio PCR reaction mixand 2 μL de-ionized water. Also present in the wells was the IPCtemplate control. Quantification reactions were carried out on AppliedBiosystems® 7500 Real-Time PCR System using the manufacture recommendedprotocol. The Applied Biosystems® 7500 Real-Time PCR System utilizes aCCD detector. The quantification results were analyzed using SDSSoftware v2.0.6 (Life Technologies). Results from this analysis areshown in FIG. 2.

Similar to what observed with the Quantifiler® Duo assay, the presenceof a filter paper punch in the reaction well results in elevatedbackground florescent signal in the FAM™ dye and VIC® dye channels. Themagnitude of the background elevation appears to be positivelycorrelated with the size of the filter paper punch in the reaction well(FIG. 2). In addition, the background florescent signal slowly increasesafter each thermal cycle and the rate of the background fluorescentsignal increase is also positively correlated to the size of the filterpaper punch (FIG. 2).

Minor Groove Binders are Responsible for the Increase in BackgroundSignal

A line of experiments was undertaken to test the role of MGBs in thebackground fluorescence in direct qrtPCR. Four probes were tested; aprobe labeled with FAM™ dye and conjugated with MGB, a probe labeledwith FAM™ dye and no MGB, a probe labeled with VIC® dye and conjugatedwith MGB and a probe labeled with VIC® dye and no MGB.

A 0.5 mm paper disc was deposited in wells of a MicroAmp® Optical96-Well Reaction Plate. Wells without punches were also analyzed. Toeach well was added 12.5 μL Quantifiler® Duo or Trio PCR reaction mixand de-ionized water along with one of each of the four primersdescribed above. Also present in the wells was the IPC template control.

Reactions were carried out on Applied Biosystems 7500 Real-Time PCRSystem using the manufacture recommended protocol. The AppliedBiosystems 7500 Real-Time PCR System utilizes a CCD detector. Thequantification results were analyzed using SDS Software v2.0.6 (LifeTechnologies). Results from this analysis are shown in FIG. 3.

As is seen in FIG. 3, background fluorescence does not increase withcycle number with probes without MGBs. In contrast, when an MGB ispresent, background fluorescence increases with cycle number.

Direct Quantification of Human DNA with and without MGBs

The experiments above identified MGBs as a source of background indirect quantification assays. These experiments were conducted in theabsence of added target and whether the presence of a target would alterthe background fluorescence had not been examined. To determine whateffect a target would have on background fluorescence and directquantification a series of experiments were conducted. In theseexperiments, punches of various diameters, 0.5 mm, 1 mm and 2 mm and 1ng. of human male DNA were placed into individual wells of MicroAmp®Optical 96-Well Reaction Plate. Wells without punches were alsoanalyzed. To each well were added 8 μL of a probe mix (with or withoutMGB), 10 μL Quantifiler® Trio PCR reaction mix and 2 μL de-ionizedwater.

Quantification reactions were carried out on Applied Biosystems® 7500Real-Time PCR System using the manufacture recommended protocol. Thequantification results were analyzed using SDS Software v2.0.6 (LifeTechnologies). Results from this analysis are shown in FIG. 4. Thepresence of a 1.0 mm or 2.0 mm paper punch affects the baseline of theamplification plot for the human male target in the presence of MGB(FIG. 4 a). All the punch sizes tested affect the baseline for the humanautosomal target when the target is detected in the presence of MGB(FIG. 4 b). In contrast, paper punch size has little effect when thehuman male or autosomal target is detected in the absence of MGB (FIGS.4 c and d). These results demonstrate that the presence or absence oftemplate does not alter the finding that the presence of MGBs isresponsible for increased background in direct quantification assays.

The Absence of Background Fluorescence is not Limited to 5′-ExonucleaseProbes

The experiments above demonstrate that MGBs are responsible forbackground fluorescence were performed using 5′-exonuclease probes; atrade name associated with these types of probes is TaqMan®. Anotherprobe type used in qrtPCR assays is a Scorpions® probe. Scorpions®probes have in a 5′ to 3′ order, a target binding region and a tailcomprising a linker and a template binding region. To investigatewhether the absence of MGBs and reduced background fluorescence waslimited to 5′-exonuclease probes a qrtPCR assay was conducted usingScorpions® probes.

The Qiagen Investigator Quantiplex HYres is a commercially availableqrtPCR assay for the quantification of total human DNA and human maleDNA. The Quantiplex HYres assay utilizes Scorpions® probes to detecttarget sequences. The probes used by the Quantiplex HYres assay do notpossess MGBs.

Punches of various diameters, 0.5 mm, 1 mm and 2 mm were made anddeposited in individual wells of a MicroAmp® Optical 96-Well ReactionPlate. Wells without punches were also included. To these wells wasadded 9 μl. of the FQ reaction mix, 9 μl. of the IC YQ primer mix and 2μl. Both the FQ and IC YQ mixes are provide as part of the InvestigatorQuantiplex HYres assay marketed by Qiagen. 2.5 ng. of human male genomicDNA was added to the wells as template. Wells without template were alsoincluded as controls.

Quantification reactions were carried out on Applied Biosystems® 7500Real-Time PCR System using the manufacture recommended protocol. Thequantification results were analyzed using SDS Software v2.0.6 (LifeTechnologies). Results from this analysis are shown in FIG. 5. Paperpunch size has little effect on the direct quantification of a humanautosomal target or Y-chromosome (FIGS. 5 a and b). Thus, the presenceor absence of increased background is not dependent on probe type butrather the presence or absence of MGBs.

I claim:
 1. A method comprising combining a fluorescently labeled probeand a filter paper contacted to a specimen in a reaction vessel,performing a quantitative real-time polymerase chain reaction (qrtPCR)and detecting a level of fluorescence emanating from the reaction vesselwith a charge coupled device during a thermal cycle while the paper isin the reaction vessel, wherein the probe is not conjugated with a minorgroove binder and the probe is a reverse complement to a multicopylocus. 2.-13. (canceled)
 14. A kit comprising a primer and afluorescently labeled probe, wherein the fluorescently labeled probecomprises a 5′ flourophore and a 3′ quencher, wherein the probe does notform a stable stem-loop structure and the probe detects a multicopylocus.
 15. The kit of claim 14 comprising at least two primers and twofluorescently labeled probes, wherein the fluorescently labeled probeseach comprise a 5′ flourophore and a 3′ quencher, wherein each of theprobes does not form a stable stem-loop structure and each of the probesdetects a different multicopy locus.
 16. The method of claim 1, furthercomprising drying the specimen on the filter paper, excising a portionof the filter paper, a dried excised filter paper, depositing the driedexcised filter paper in the reaction vessel, the dried excised filterpaper not being contacted to a liquid until the qrtPCR assay.
 17. Themethod of claim 1, wherein a linear scale graph of the level offluorescence emanating from the reaction vessel as expressed as ΔRnversus cycle number produces a sigmoid curve.